The present invention relates to a biosensor for measuring the concentration of one or more specific substances contained in one or more sample solutions and a measuring method using the biosensor.
As a conventional biosensor for measuring the concentrations of a plurality of specific substances simultaneously, there is a flow injection type biosensor. One such example is Flow Injection Analyzer (FIA) YSI MODEL 2700 SELECT (Yellow Spring Instrument Co., Inc.), which is a biosensor utilizing an immobilized enzyme membrane and electrode reaction, and its operation for simultaneously measuring glucose and L-lactic acid contained in a sample solution is described below. First, the tip end of a sample suction tube is immersed in a sample solution to suck the sample solution into a measuring system, whereby the sample solution is supplied into a sample chamber which is a measuring site. At the same time, a certain amount of buffer solution is also introduced into the sample chamber, where the sample solution and the buffer solution are stirred and mixed with each other by a stirrer of the sample chamber. The sample chamber is furnished with two electrodes having an immobilized enzyme membrane of glucose oxidase (hereinafter referred to as GOx) and an immobilized enzyme membrane of lactate oxidase (hereinafter referred to as LOD), respectively. After a lapse of a predetermined time period from the supply of the sample solution and the buffer solution, an electrochemical measurement is performed to calculate the concentrations of glucose and L-lactic acid contained in the sample solution.
Also, an example of the biosensor for measuring a single specific substance in a plurality of sample solutions successively is Glucoroder GTX (A&T Co., Inc.), which measures the concentration of glucose in blood, and its measuring operation is described below. A certain amount of plasma, which is a sample solution, is filled in the sample cups of a circular fraction collector, and the sample cups are set to the fraction collector to start a measurement. First, an aspirator automatically sucks a certain amount of the sample solution from the first sample cup on the fraction collector and moves to a measuring chamber containing a buffer solution, where the sample solution is diluted with the buffer solution by a predetermined dilution factor. Subsequently, an immobilized enzyme electrode with an immobilized GOx membrane is immersed in the sample solution in the measuring chamber, and after a lapse of a predetermined time period, an electrochemical measurement is performed. When the measurement of the first sample cup is completed, the fraction collector turns so that the second sample cup moves to the position of the first sample cup, and the sample solution of the second sample cup is measured. In this way, upon completion of the measurement of one sample cup, the fraction collector turns so that another sample cup moves to the position where the aspirator can operate, and measurements can be performed successively.
Meanwhile, as a system for measuring a plurality of specific substances in a sample solution without dilution or stirring with ease, the following biosensor has been proposed in Japanese Laid-Open Patent Publication No. Hei 5-196596. This biosensor has electrode systems on opposite sides of the insulating base plate, one on each side. On each of the electrode systems, a reaction layer having a different enzyme or different combination of enzymes is formed directly or indirectly. A method of measuring glucose and fructose in a sample solution using this biosensor is described below. First, an electrode system is formed by screen printing on each side of the insulating base plate. A reaction layer containing GOx is formed on the electrode system on one side of the base plate, while a reaction layer containing fructose dehydrogenase (hereinafter referred to as FDH) is formed on the electrode system on the other side of the base plate. Further, a cover member is joined to each side of the base plate to fabricate a sensor. The cover member forms a sample solution supply pathway, through which a sample solution is introduced into the electrode system, between itself and the base plate. The enzyme reaction time of GOx is shorter than that of FDH. Thus, by applying a voltage to the electrode systems after one minute and two minutes, respectively, from the simultaneous supply of the sample solution to the respective sample supply inlets of the sensor, and measuring the current five seconds later, the concentrations of glucose and fructose in the sample solution can be quantified.
The above-described conventional biosensors of flow injection type for measuring the concentrations of a plurality of specific substances simultaneously need a buffer solution serving as a carrier for making a measurement. In the above example, since the buffer solutions of GOx and LOD are the same, the two components can be measured simultaneously. However, if the appropriate pHs of the enzymes for the plurality of specific substances to be measured or the appropriate kinds of buffer solutions are different, simultaneous measurement is impossible. Also, the measuring system becomes large-scale, and the maintenance becomes complicated.
Also, the above-described conventional biosensor for measuring a single specific substance contained in a plurality of sample solutions needs a large-scale means, such as the fraction collector, for supplying samples successively. Further, since simultaneous measurement is not possible, the sample solutions evaporate with passage of time, which may result in a decreased measuring accuracy.
Further, the above-described biosensor having a plurality of electrode systems on the base plate is unable to divide the current of the plurality of electrode systems and detect the current of each electrode system in the case where there is no difference in enzyme reaction time among the enzymes and combination of enzymes contained in a plurality of reaction layers. Thus, when there is no difference in enzyme reaction time among the enzymes for a plurality of specific substances to be measured and when the specific substance to be measured is one, measurement is not possible.
In view of these problems, an object of the present invention is to provide a biosensor having a simple structure and capable of measuring the concentrations of a plurality of specific substances simultaneously even in the cases where the appropriate pHs of the enzymes for the plurality of specific substances to be measured or the appropriate kinds of buffer solutions are different and there is no difference in enzyme reaction time among the enzymes.
Another object of the present invention is to provide a biosensor having a simple structure and capable of measuring a single specific substance contained in a plurality of sample solutions continuously with high accuracy.
Further object of the present invention is to provide a measuring method using the biosensor.